Dataset Information

Translational Read-Through Therapy of RPGR Nonsense Mutations.

ABSTRACT: X-chromosomal retinitis pigmentosa (RP) frequently is caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We evaluated the potential of PTC124 (Ataluren, Translama^TM) treatment to promote ribosomal read-through of premature termination codons (PTC) in RPGR. Expression constructs in HEK293T cells showed that the efficacy of read-through reagents is higher for UGA than UAA PTCs. We identified the novel hemizygous nonsense mutation c.1154T > A, p.Leu385* (NM_000328.3) causing a UAA PTC in RPGR and generated patient-derived fibroblasts. Immunocytochemistry of serum-starved control fibroblasts showed the RPGR protein in a dot-like expression pattern along the primary cilium. In contrast, RPGR was no longer detectable at the primary cilium in patient-derived cells. Applying PTC124 restored RPGR at the cilium in approximately 8% of patient-derived cells. RT-PCR and Western blot assays verified the pathogenic mechanisms underlying the nonsense variant. Immunofluorescence stainings confirmed the successful PTC124 treatment. Our results showed for the first time that PTC124 induces read-through of PTCs in RPGR and restores the localization of the RPGR protein at the primary cilium in patient-derived cells. These results may provide a promising new treatment option for patients suffering from nonsense mutations in RPGR or other genetic diseases.

SUBMITTER: Vossing C

PROVIDER: S-EPMC7697989 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Translational Read-Through Therapy of RPGR Nonsense Mutations.

Vössing Christine C Owczarek-Lipska Marta M Nagel-Wolfrum Kerstin K Reiff Charlotte C Jüschke Christoph C Neidhardt John J

International journal of molecular sciences 20201110 22

X-chromosomal retinitis pigmentosa (RP) frequently is caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We evaluated the potential of PTC124 (Ataluren, TranslamaTM) treatment to promote ribosomal read-through of premature termination codons (PTC) in RPGR. Expression constructs in HEK293T cells showed that the efficacy of read-through reagents is higher for UGA than UAA PTCs. We identified the novel hemizygous nonsense mutation c.1154T > A, p.L ...[more]

PMID: 33182541

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Project description:Premature-termination codons (PTCs) in CFTR (cystic fibrosis [CF] transmembrane conductance regulator) result in nonfunctional CFTR protein and are the proximate cause of ∼11% of CF-causing alleles, for which no treatments exist. The CFTR corrector lumacaftor and the potentiator ivacaftor improve CFTR function with terminal PTC mutations and enhance the effect of readthrough agents. Novel correctors GLPG2222 (corrector 1 [C1]), GLPG3221 (corrector 2 [C2]), and potentiator GLPG1837 compare favorably with lumacaftor and ivacaftor in vitro. Here, we evaluated the effect of correctors C1a and C2a (derivatives of C1 and C2) and GLPG1837 alone or in combination with the readthrough compound G418 on CFTR function using heterologous Fischer rat thyroid (FRT) cells, the genetically engineered human bronchial epithelial (HBE) 16HBE14o- cell lines, and primary human cells with PTC mutations. In FRT lines pretreated with G418, GLPG1837 elicited dose-dependent increases in CFTR activity that exceeded those from ivacaftor in FRT-W1282X and FRT-R1162X cells. A three-mechanism strategy consisting of G418, GLPG1837, and two correctors (C1a + C2a) yielded the greatest functional improvements in FRT and 16HBE14o- PTC variants, noting that correction and potentiation without readthrough was sufficient to stimulate CFTR activity for W1282X cells. GLPG1837 + C1a + C2a restored substantial function in G542X/F508del HBE cells and restored even more function for W1282X/F508del cells, largely because of the corrector/potentiator effect, with no additional benefit from G418. In G542X/R553X or R1162X/R1162X organoids, enhanced forskolin-induced swelling was observed with G418 + GLPG1837 + C1a + C2a, although GLPG1837 + C1a + C2a alone was sufficient to improve forskolin-induced swelling in W1282X/W1282X organoids. Combination of CFTR correctors, potentiators, and readthrough compounds augments the functional repair of CFTR nonsense mutations, indicating the potential for novel correctors and potentiators to restore function to truncated W1282X CFTR.

Dataset Information

Translational Read-Through Therapy of RPGR Nonsense Mutations.

Publications

Translational Read-Through Therapy of <i>RPGR</i> Nonsense Mutations.

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